Leptin, an adipocyte-derived hormone, delivers its appetite-suppressing signals by passing the blood-brain barrier and binding to the specific signaling form of its receptor in certain nuclei of the hypothalamus. In this way, leptin constitutes a feedback mechanism regulating adipose tissue mass. Mutations within the leptin system result in a marked obese phenotype and impaired endocrinological functioning. (Zhang et al., 1994; Chen et al., 1996.) Although the long signaling form of the leptin receptor shows high expression levels in these hypothalamic nuclei, it is also expressed in several peripheral tissues including lung, liver, lymph nodes and gonads. (Tartaglia et al., 1995; Ghilardi et al., 1996.) This leads to the involvement of leptin in several peripheral functions and makes a typical pleiotropic cytokine.
Until today, the therapeutic use of leptin as a weight-reducing agent was limited. (Heymsfield et al., 1999.) It is observed that in most obese people, a strong correlation exists between adipose mass and leptin levels, a phenomenon often explained by leptin resistance. (Maffei et al., 1995.) A number of possible explanations for this resistance have been suggested and include: a saturable transport through the blood-brain barrier resulting in a limited leptin activity in the hypothalamus (Schwartz et al., 1996; E l Haschimi et al., 2000), cross-talk with the glucocorticoid system (Zakrzewska et al., 1997), or defects at the leptin receptor level, such as elevated expression of the signaling inhibitor SOCS3 (Bjorbaek et al., 1998).
Recently, it became clear that leptin not only plays a role in regulating food intake, but also functions in angiogenesis. (Sierra-Honigmann et al., 1998.) Further, leptin functioning in invasiveness of kidney and colonic epithelial cells (Attoub et al., 2000) have been demonstrated.
Being a member of the type 1 cytokine receptor family, the leptin receptor is activated by cross-phosphorylation of associated JAK kinases, most likely JAK2 and/or JAK1. (Bjorbaek et al., 1997; Banks et al., 2000.) Activation of the leptin receptor leads to recruitment of signaling molecules containing phosphotyrosine-binding SH2 molecules. Signal Transducers and Activators of Transcription (STAT) molecules (Baumann et al., 1996; Vaisse et al., 1996) and the receptor-associated SH2-containing phosphatase SHP-2 (Carpenter et al., 1998; Li and Friedman, 1999) are recruited in the activated leptin receptor complex. Leptin-mediated activation of Mitogen-Activated Protein Kinases (MAPK) and Insulin Receptor Substrate 1 (IRS-1) have been shown in various cell systems. (Cohen et al., 1996; Bjorbaek et al., 1997; Takahashi et al., 1997; Banks et al., 2000.) Promotion of invasiveness by leptin seems to be mediated by phosphoinositide 3-kinase, Rho- and Rac-dependent signaling pathways. (Attoub et al., 2000.)
Leptin rapidly induces Cytokine-Inducible SH2-containing protein (CIS), both in vitro and in vivo. Leptin can also strongly and rapidly induce the production of the signal transduction inhibitor Suppressor of Cytokine Signaling 3 (SOCS3) in various cell types and in vivo. (Bjorbaek et al., 1998; Emilsson et al., 1999; Waelput et al., 2000.) Both CIS and SOCS3 are members of an expanding family of SH2 containing proteins which are typically built up of a pre-SH2 domain, a central SH2 domain and a highly conserved SOCS box sequence. The latter motif is also found in a number of other signaling molecules (Hilton et al., 1998) and seems to be connected with proteasome function. (Zhang et al., 1999.) The observation that the leptin-resistant Ay/a mutant mice strain shows elevated SOCS3 levels makes this protein a possible mediator of leptin resistance. (Bjorbaek et al., 1998.)
Recently, it has been shown that a tyrosine recruitment site within gp130, the signaling component of the IL-6 complex, is required for binding and, thus, for the inhibitory activity of SOCS3. (Nicholson et al., 2000; Schmidt et al., 2000.) This is in contrast to SOCS1, which binds directly to JAK kinases and directly inhibits their kinase activity. (Yasukawa et al., 1999.) Similar results have been obtained for the insulin receptor and the erythropoietin receptor. (Emanuelli et al., 2000; Sasaki et al., 2000.)
In previous studies, it has been shown that leptin induces two gene sets in the PC12 rat pheochromocytoma cell line stably expressing the mouse leptin receptor. Many of these genes appear to be regulated in vivo. (Waelput et al., 2000.) Using a mutational approach, it was shown that in the mouse leptin receptor residue Y985 is involved in a negative feedback signal, and furthermore, that this effect is more pronounced when mutated in concert with another tyrosine, Y1077. (Eyckerman et al., 1999.) However, phosphorylation of this tyrosine has never been demonstrated, suggesting that this site was playing only a minor role. Surprisingly, a short functional fragment around the Y1077 was found to be sufficient for SOCS3 and CIS binding and/or signaling. Moreover, it was demonstrated that the functional fragment is also sufficient for Vav signaling. Vav seems to be a general signaling molecule, but has never been shown to be involved in leptin signaling. The short functional sequence is extremely conserved, which makes it an attractive target for pharmaceutical compositions modulating SOCS3, CIS and/or Vav-mediated signaling in general, and leptin-induced signaling in particular.